Removal of the outer cell wall layers of a gram-negative marine pseudomonad (B-16) showed that these cells are penetrable by actinomycin D and that, therefore, neither the cytoplasmic membrane nor the peptidoglycan layer constitutes the barrier which excludes this antibiotic from intact cells, but that this barrier is formed by the outer layers of the cell wall which include the lipopolysaccharide component and the double-track layer.